The molecular toxicology of Amanita intoxication is not well understood. Two toxic peptides alpha-amanitin and phalloidin, have been isolated and their structures determined, but the mechanism of action of only one of these is known. It has been shown that alpha-amanitin inhibits the form II (nucleoplasmic) DNA-dependent RNA polymerase in vitro, but the effect in vivo is less well understood. There is no known single effect of phalloidin. It is therefore proposed to study the effect of alpha-amanitin in vivo and to begin a rational search for the mechanism of action of phalloidin. The experimental rationale will examine the mechanism of action of alpha-amanitin in vivo by its effect on nucleolar structure and function. It is proposed that nucleolar structure (and therefore function) is under direct or indirect control by the product of the form II RNA polymerase. That is, nucleolar structure is dependent upon a unique specie(s) of RNA product of the form II enzyme or a specific (rapidly turning over?) protein translated from this messenger RNA. Specifically, the effect of alpha-amanitin on nucleolar protein turnover will be examined using labeled amino acids and analysis of nucleolar proteins by polyacrylamide gel electrophoresis. Synthesis of RNA (ribosomal and messenger) will be studied using labeled uridine or orotic acid. Analysis of RNA will be made on sucrose density gradients and polyacrylamide gel electrophoresis. Polyribosomes will be examined also on sucrose gradients. Correlation of this proposed mechanism of action of alpha-amanitin to the natural events in the cell cycle will be attempted using synchronous tissue culture cells to relate the control of nucleolar structure to function. Phalloidin will be studied in vivo and in vitro to define its mechanism of action. Autoradiography will be performed to relate the subcellular location to toxic action. Phalloidin or its metabolite(s) will be identified and studied employing the labeled compound. The effect on protein synthesis will be studied in reticulocytes and its suggested effects on mitochondria re-examined.